Predicting cancer progression

ABSTRACT

Method of selecting treatment for a subject diagnosed with cancer comprises contacting antibody that binds specifically to thymidine kinase 1 (TK1) protein with a first body fluid sample before completing cancer treatment; determining a first amount of antibody binding to TK1 protein in the first sample; correlating the first amount to a first concentration of TK1 protein using a standard curve correlating an amount of antibody binding to recombinant human TK1 (rhTK1) and a concentration of rhTK1; contacting the antibody with a second body fluid sample within one to six months after the cancer treatment; determining a second amount of antibody binding to TK1 protein in the second sample; correlating the second amount to a second concentration using the curve; identifying the patient as having a high risk of future cancer relapse if the second concentration is ≥ the first concentration; and selecting an adjuvant treatment based on the identification.

The Sequence Listing entitled “Dec-30-2019-Sequence-Listing_ST25.txt”created Dec. 30, 2019, having a size of 3530 bytes, is incorporatedherein by reference.

TECHNICAL FIELD

The present invention generally refers to monitoring and prediction ofcancer diseases and in particular to early prediction of progression andrecurrence of cancer diseases.

BACKGROUND

Cancer is a leading cause of death in humans and the number of affectedindividuals increases for each year. Although the different methods oftreatment for cancer, e.g. chemotherapy, endocrine therapy, radiotherapyand surgery, have improved tremendously the last decades, they are farfrom perfect, in particular for patients with late stages of cancer.Thus, much research has been spent on early detection of tumors inpatients.

One used method for tumor and tumor stage detection is determination ofcell proliferation in patients. The proportion of DNA-synthesizing cells(S-phase cells) in tumors has been used as a measure of theirproliferation rate. The DNA-synthesizing cells have earlier beendetermined by means of radioactive-labeled thymidine (autoradiography).Incorporation of halogen-analogs of thymidine (BrdU) and antibodiesagainst these have been used together with quantitative flow-cytometricDNA measurements. The disadvantage with the use of isotopic labeledthymidine and BrdU is that only living cells can be measured. Therefore,these methods have only been used on selected patients and in a limitednumber of studies [Wilson, Acta Oncol., 30:903, 1991]. Furthermore, theflow-cytometry technique is unable to distinguish between proliferatingand non-proliferating cells. Determination of S-phase cells is even morecomplicated in tumors, since the cancer tumor cells do not deviate intheir DNA-content from benign cells [Tribukait, World J. Urol., 5:108,1987].

Instead of measuring DNA-synthesis itself, markers related toproliferating cells has been used, for example, Ki-67 and PCNA(Proliferating Cell Nuclear Antigen). Ki-67 is expressed in all cellcycle stages except for GO [Scholzen, J. Cellular Physiol., 182:311,2000]. Antibodies against Ki-67 can be used on fresh tissues as well ason formaline fixed and paraffin embedded tissues. Depending on the cellproliferation rate, PCNA is expressed in all cell cycle stages. PCNA isnot as sensitive for various fixation techniques as Ki-67 [Hall, CellTissue Kinet., 25:502, 1990]. Antibodies against other types of proteinsand enzymes involved in the DNA-synthesis (DNA polymerase,ribonucleotide reductase) have been tested, but with limited successfulresults [Wilson, Acta Oncol., 30:903, 1991].

Thymidine kinase (TK), an enzyme of the pyrimidine salvage pathway,catalyses the phosphorylation of thymidine to thymidine monophosphate.TK in human cells appears in two forms, a cytoplasmic (TK1) and amitochondrial (TK2) protein, encoded by different genes. Human TK1 andTK2 are located on the chromosome 17q23.2-q25.3 and 16q22-q23.1,respectively. TK1 transcripts encode a 25.5 kDa protein with highlyconserved regions typical for nucleoside kinases. However, the crystalstructures of this enzyme family have no yet been determined. Theexpression of TK1 is cell cycle regulated and the TK1 regulation iscomplex with mRNA levels peaking in proliferating cells. Splicing andtranslation of TK1 mRNA also varies in cells at different growth states.TK1 levels are mainly regulated by post-translational mechanisms, inparticular by differential degradation due to highly active proteaseexpression in mitotic cells. The C-terminal region of TK1 contains aspecific sequence, KEN, which recently has been shown to be the signalfor mitotic degradation of TK1 by the Anaphase-promotingcomplex/cyclosome-Cdh1-mediated pathway [Ke, Mol. Cell. Biol., 24:514,2004]. TK2 is not cell cycle regulated and is the only TK enzyme foundin resting cells [Wintersberger, Biochem. Soc. Trans., 25:303, 1997;Sherley, J. Biol. Chem., 263:8350, 1988; He, Cell. Prolif., 24:3, 1991;Kauffman, Mol. Cell. Biol., 11:2538, 1991; Hengstschläger, J. Biol.Chem., 269:13836, 1994; Munch-Petersen, J. Biol. Chem., 266:9032, 1991].

The majority of the above-mentioned markers have been used to identifyproliferation cells in tissues. However, thymidine kinase activity hasbeen determined in cytosol fractions of tissues as a proliferationmarker in human breast cancer. In a study of 1,692 breast cancer patient[Broet, J. Clin. Oncol., 19:2778, 2000], high TK1 activity in thecytosol correlated to a shorter survival as well as a poor outcome ofendocrine treatment (tamoxifen) [Foekens, Cancer Res., 61:1421, 2001].Furthermore, thymidine kinase has also been used as a marker of cellproliferation in serum by measuring its enzyme activity.

Because of the tight coupling between the serum TK (STK) enzyme activityand high proliferation, it is considered a sensitive and useful markerfor cell proliferation and hence for malignancy detection [He, Internal.J. Biol. Marker, 15:139, 2000; Zou, Internal. J. Biol. Marker, 17:135,2002; He, Biochimica Biophysica Acta, 1289:25, 1996; He, Europ. J. CellBiol., 70:117, 1996; Wu, Anticancer Res., 6:4867, 2000; Mao, CancerInves. 20:922, 2002; Wang, Analysis Cell. Pathology, 23:11, 2001;Kuroiwa, J. Immuno. Methods, 253: 1, 2001]. Thus, STK enzyme activityhas been used as a tumor marker in patients with different blood tumors.However, STK activity has been found to be not a good marker in patientswith solid tumors.

More than 95% of STK enzyme activity corresponds to TK1 while less than5% corresponds to TK2. The composition and the properties of STK are notyet well understood. Results indicate that STK is a polymeric form ofTK1, probably in complex also with other serum proteins and it has atotal molecular weight of approximately 700 kDa [Karlström, Mol. Cell.Biochem, 92:23, 1990].

Using the thymidine analogue 5-iodo-2′-deoxyuridine as substrate, STKactivity was established as a serological proliferation marker in 1984[Gronowich, Br. J. Cancer, 47:487, 1983] and is now available as acommercial RIA-kit-[¹²⁵I]-radio-assay (Sangtec Medical AB, Stockholm,Sweden, recently purchased by DiaSorin Inc.). The STK activity assay hasbeen useful for estimation of tumor spread and prognosis in patientswith the acute leukemia and chronic leukemia (CLL), Hodgkin andnon-Hodgkin's lymphoma, but not in the case of solid tumors [Gronowich,Int. J. Cancer, 15:5, 1984]. The STK enzyme activity in CLL patientprovides useful prognostic information regarding both responses totherapy and length of survival. Although the method is relativelyeffective, especially for leukemia and lymphoma diseases, the5-iodo-2′-deoxyuridine is not a specific substrate for STK (TK1)activity. Furthermore, the [¹²⁵I]-radio-iodo-deoxyuridine has a shorthalf-life (four weeks), the STK enzyme activity is highly sensitive totemperature and pH changes, and the radioassay requires specializedequipment (γ-scintillation counter), isotope laboratory and highlyskilled personnel. The disadvantages of such a radioassay technique haveprobably limited the clinical use of this assay.

Therefore, a new STK activity kit based on antibodies against theproduct of the STK reaction has recently been developed. Anti-TK1antibodies have also been generated during the last decades, mostlypolyclonal antibodies for basic research and not for commercial use.Recently, mouse mono- and polyclonal anti-TK1 antibodies have beendeveloped for potential clinical purpose, i.e. for breast cancer [Mang,Cancer Detection Prev., 25:8, 2001] and lung cancer [Voeller,Anti-Cancer Drugs, 12:555, 2001]. One anti-TK1 monoclonal antibody isnow commercial available for basic research, but not for clinical use(QED Bioscience Inc, San Diego, USA, 2003).

In the U.S. Pat. No. 5,698,409 O'Neil discloses monoclonal antibodies(mAb) generated against the enzymatically active 100 kDa tetrameric formof TK1 from Raji cells. The mAb are used for determining only thisenzymatically active form of TK1 in biological samples from patients.Furthermore, O'Neil has to determine the TK activity using radiolabledthymidine, in addition to using the mAb. The determined TK1 activitiesare then used for diagnosing and monitoring various forms of cancer. Theproperties of their anti-TK1 monoclonal antibodies raise questions aboutthe specificity of these antibodies, i.e. they react with a protein ofhigher molecular weight than expected in SDS gelelectrophoresis Westernblot (45 kDa versus 25 kDa in extracts of human HeLa cells).Furthermore, the antibodies detect a protein in the serum occurring inanother (higher) concentration range than found by other workers in thefield, i.e. they need to dilute serum 16,000 times while others useundiluted serum. These facts suggest that their antibodies react withother proteins in addition to enzymatically active TK1 and/or TK2. Inaddition, the method of O'Neil is impaired with the similar problems aswith usage of the radiolabled thymidine analogue discussed above.

Kuroiwa, et al, [Kuriowa, J. Immuno. Methods, 253:1, 2001] havedeveloped and tested 26 anti-TK1 monoclonal antibodies. The propertiesof these antibodies are more as expected, i.e. they react with a proteinwith a molecular weight of 25 kDa in extracts of human HeLa in Westernblot with no reported cross-reactivity to other proteins. When they usedthese antibodies in serum from patients with solid tumors using ELISA,they found no significant differences of serum TK1 levels as compared toserum TK1 levels from healthy individuals.

SUMMARY

It is a general object of the present invention to provide a test forearly prediction of progression and relapse of a cancer disease in asubject.

It is another object of the invention to provide a decision supportmethod for estimating the likelihood of progress of a cancer disease ina subject.

It is a particular object of the present invention to provide earlyprediction of local and/or metastatic appearance or recurrence of tumorsin a subject following treatment of cancer.

These and other objects are met by the invention as defined by theaccompanying patent claims.

Briefly, the present invention involves early prediction of, orestimation of the likelihood of, progression and relapse of a cancerdisease in a subject diagnosed and possibly treated for cancer. Theinvention is based on determining a binding response level of animmunoreactive material (IM) comprising thymidine kinase 1 (TK1) proteinin a sample, preferably a body fluid sample, e.g. blood serum sample,from a subject, preferably mammalian subject and more preferably humansubject, at cancer diagnosis and/or after cancer treatment (e.g. afteroperation, chemotherapy, endocrine therapy radiotherapy and/orimmunological therapy treatment). The likelihood of tumor recurrence isthen estimated based directly on this determined IM binding responselevel.

The determined IM binding response level may in one embodiment bedetermined for the subject at multiple time occasions. Preferably, afirst taking of sample and binding response determination is prosecutedno later than one month after initiation of the cancer treatment, morepreferably before completion of the treatment, such as before startingthe cancer treatment in the subject. A second sample is preferably takenfrom the subject and analyzed for IM binding response level afterstarting the treatment, such as within the first year following thecancer treatment. More preferably, the second IM binding response levelis determined in the subject between one month up to one year, such asbetween one month and six months, or around 3 months, after thetreatment has begun or is completed. In either case, the second IMbinding response level is preferably not determined until at least oneweek, e.g. some weeks, one month, or multiple months, have elapsed sincethe determination of the first IM binding response level in the samesubject.

The prediction of progress of the cancer disease is then based on acomparison of the two IM binding response levels or by investigation ofa quantity derived therefrom. For example, the subject has a highlikelihood of tumor relapse if the second IM binding response level isequal to or exceeds the first IM binding response level, or expressed inanother way, the likelihood of tumor relapse is high if the ratio of thesecond (subsequent) IM binding response level and the first IM bindingresponse level exceeds, or is equal to, one.

Alternatively, the determined IM binding response level in the subjectcould compared to a normal IM binding response level, as determined frombody fluid samples of healthy subjects, whereby local and/or metastatictumor appearance is predicted based on this comparison.

The IM binding response level is preferably determined by contacting thebody fluid (e.g. serum) sample with a ligand that has affinity for theTK1 protein. In a preferred embodiment of the invention the ligand is anantibody that binds specifically to a surface exposed epitope of the TK1protein, preferably to a defined immunogenic sequence of the C-terminalpart of TK1 protein. This ligand should preferably be able to detectboth enzymatically active and inactive forms of the TK1 protein andpossible also the TK1 protein complexed with other macromolecules. Theamount of antibody binding is then measured by a suitable and sensitiveassay method, preferably the enhanced chemoluminescence (ECL) dot blotassay system and more preferably such an ECL dot blot assay system usingnitrocellulose membranes.

From this measured IM binding response level a concentration of IM canbe determined using a standard, e.g. recombinant human TK1 (rhTK1). Insuch a case, the binding response using an anti-TK1 antibody is measuredin samples with different known concentrations of rhTK1 and a standardcurve of binding response vs. concentration may be generated. Asubsequent IM binding response level measurement from a subject treatedfor cancer can then be related to a corresponding IM concentration usingthe standard curve. The estimation of the likelihood of cancerprogression and recurrence is then based directly on this IMconcentration.

This means that in the present invention, the relapse of a cancerdisease in a subject treated for the disease is determined based on thebinding response level or concentration level of the TK1 proteincomprising immunoreactive material. This is in clear contrast to theprior art techniques that use the thymidine kinase activity for tumorrecurrence prediction.

By determining the IM binding response level in a patient within thefirst few months after cancer diagnosis and treatment, it is possible todetect and identify those patients that run a high risk of a subsequent(within 1 to 3 years, possibly up to 10 years, after the treatment)cancer disease relapse. Thus, the invention allows early detection ofthose patients that have a high and low risks of tumor relapse, whichenables differential treatments and increases the chances of success ofthe treatment and may prolong survival of the patient, but also avoidsinefficient and thus unnecessary treatments.

The TK1 protein comprising immunoreactive material includes the TK1protein, e.g. serum TK1 protein, an ezymatically active and/or inactivemonomeric and/or polymeric (e.g. dimeric, tetrameric) form of the TK1protein and/or the TK1 protein complexed with other molecules, e.g.proteins, such as inhibitors.

The invention may be applicable to most types of cancers and isespecially adapted for cancers with solid tumors, such as breast cancer,gastric cancer and lung cancer.

The invention offers the following advantages:

-   -   Early detection of treated cancer patients running a risk of        tumor relapses;    -   Provides decision support allowing differential cancer        treatment, avoiding over-treatment, and/or change of treatment        strategy; and    -   Enables increased survival chances and quality of life of cancer        patients.

Other advantages offered by the present invention will be appreciatedupon reading of the below description of the embodiments of theinvention.

SHORT DESCRIPTION OF THE DRAWINGS

The invention together with further objects and advantages thereof, maybest be understood by making reference to the following descriptiontaken together with the accompanying drawings, in which:

FIG. 1 illustrates correlation between enzyme activity of serumthymidine kinase (STK) and concentration of TK1 protein comprisingimmunoreactive material (IM) (STK1) in healthy individuals;

FIG. 2 illustrates IM concentration (STK1) in serum of healthyindividuals and human patients with different types of tumors, where Arepresents leukemias, B represents gastric tumors, C represents colontumors, D represents rectum tumors, E represents breast tumors, Frepresents lung tumors, G represents lymphomas, H represents hepatomas,I represents brain tumors, J represents other types of malignant tumors,K represents morphological benign lesions and L represents healthypersons;

FIG. 3 illustrates IM concentration (STK1) measured by Western blot anddot blot from a patient with a gastric cancer (P3), a dot blot of ahealthy individual and of human recombinant TK1;

FIG. 4 illustrates correlation between IM concentration (STK1) andenzyme activity of STK in patients with benign lesions and malignanttumors before cancer treatment, where A represents leukemias, Brepresents gastric tumors, C represents colon tumors, D representsrectum tumors, E represents lung tumors, F represents lymphomas, Grepresents hepatomas, H represents brain, I represents morphologicalbenign lesions tumors and J represents breast tumors;

FIG. 5 illustrates the cumulative incidence of any recurrence in 67breast cancer patients, divided into three equal groups representingrelative STK1 concentrations of <0.78, 0.78-1.08 and >1.08, as definedby the STK1 concentration determined at three months after surgerydivided by the corresponding STK1 concentration at 21 days aftersurgery;

FIG. 6 is a comparison of IM concentration (STK1), enzyme activity ofSTK and concentration of CA 15-3 in serum at 3 months after operation in37 breast cancer patients with (A) and without (B) subsequent cancertumor relapse expressed in percentage of measured values 21 days afteroperation; and

FIG. 7 is an illustration of a flow diagram of the tumor relapsepredicting method of the present invention.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich the present invention belongs. For clarity of the invention, thefollowing definition is used herein.

“Thymidine kinase” (ATP: thymidine-5′ phosphotransferase, EC.2.7.1.21 inthe International Union of Biochemistry classification system) is anenzyme of the pyrimidine salvage pathway and catalyses thephosphorylation of thymidine to thymidine monophosphate. In human cells,TK appears in two forms, a cytoplasmic thymidine kinase 1 (TK1) and amitochondrial thymidine kinase 2 (TK2) form, encoded by different genes.“Enzyme activity of TK” refers to the roll of TK in catalyzing thephosphorylation of thymidine to thymidine monophosphate.

“TK1 protein comprising immunoreactive material (IM)” refers to amaterial or composition comprising the TK1 protein. The IM materialcould comprise both enzymatically active and inactive forms of the TK1protein, including e.g. monomeric, polymeric (e.g. dimeric, tetrameric)TK1 protein forms and/or all forms of the TK1 protein. The material canalso or instead include the (enzymatically active or inactive) TK1protein complexed with other molecules, including proteins andpolypeptides, such as inhibitors and/or activators.

According to an aspect of the present invention there is provided amethod of early prediction of progression and relapse or recurrence of acancer disease in a subject, preferably a mammalian subject and morepreferably a human subject, treated for cancer. The method comprisesdetermination of a binding response level of an immunoreactive material(IM) comprising the thymidine kinase 1 (TK1) protein in a sample fromthe subject. The likelihood of progress of the cancer disease, e.g. asmanifested in appearance or recurrence a cancer tumor, is then estimatedbased directly on the determined IM binding response level.

In a particular aspect of the invention the binding response level isdetermined in an immunoreactive material that includes bothenzymatically active and inactive forms of the TK1 protein. In anembodiment of the invention, this binding response level is expressed asa concentration level of IM in a sample from a subject using a ligandthat has affinity for TK1. The likelihood of progression and relapse ofthe cancer is then estimated based directly on the determined IMconcentration level.

In an embodiment of the invention the IM binding response level isdetermined for the subject at multiple, i.e. at least two, timeoccasions. The likelihood of disease relapse is then based on acomparison of these at least two binding response levels. In a preferredembodiment of the invention, the first taking of sample and bindingresponse determination is prosecuted no later than one month afterinitiation of the cancer treatment, more preferably before completion ofthe treatment, such as before starting the cancer treatment in thesubject or in connection of diagnosis of the cancer disease. The atleast second sample is preferably taken from the subject and analyzedfor IM binding response level after starting the treatment, such aswithin the first year following the cancer treatment. More preferably,the second IM binding response level is determined in the subjectbetween one month up to one year, such as between one month and sixmonths, or around 3 months, after the treatment has begun or iscompleted. In either case, the second IM binding response level ispreferably not determined until at least one week, e.g. some weeks, onemonth, or multiple months, has elapsed since the determination of thefirst IM binding response level in the same subject.

The prediction of progress of the cancer disease and/or tumor recurrenceis then based on a comparison of the two IM binding response levels orby investigation of a quantity derived therefrom. For example, thesubject has a high likelihood of cancer relapse if the second IM bindingresponse level is equal to or exceeds the first IM binding responselevel, or expressed in another way, the likelihood of tumor relapse ishigh if the ratio of the second (subsequent) IM binding response leveland the first IM binding response level exceeds, or is equal to, one.

The IM binding response level in the subject is preferably periodicallyor intermittently determined at several time occasions during and afterthe cancer treatment, e.g. after operation, after start of chemotherapytreatment, after start of endocrine therapy treatment, after start ofradiotherapy treatment and/or after start of immunological therapytreatment, in the patient. Thus, the IM level is preferably determinedduring the first next years, e.g. up to the next 5 to 10 years,following treatment to, as early as possible, detect progress of thecancer disease in the subject. However, for most cancer types, andespecially solid tumor cancer types, the method of the invention isprobably able to detect any subsequent cancer relapse within the first 6months, such as within the first 3 months, following the cancertreatment. These subsequent binding response levels are preferablycompared to corresponding binding response levels determined earlier forthe same subject, e.g. the first IM binding response level discussedabove.

In other words, the present invention is able to predict within thefirst few months (typically 0-6 months, for some cancers 0-3 months)following cancer treatment whether a subject will anew get (local and/ormetastatic) tumors back. Thus, although a cancer disease will reappearfirst within a couple of years after the treatment, the invention maypredict its progression and relapse within a few months after thetreatment. By means of such an early prediction, additional treatmentand/or change of treatment strategy may be applied in order to preventthe predicted local and/or metastatic tumor appearance. In addition,those subjects that are predicted to be likely to get a relapse may beunder close surveillance to, as early as possible, detect any newtumors, and thus increase the survival chances of the subject.Identification of low risk patients with the same assay would alsoimprove the qualitative of patients' life by reducing the risk ofover-treatment or unnecessary treatments.

In another embodiment of the invention, the determined IM bindingresponse level is compared to a normal binding response level, asdetermined from healthy tumor-free subjects. The estimation of thelikelihood of cancer progression and relapse is then based on thiscomparison. For example, the disease is likely to progress and relapseif the determined IM level is significant higher than the normal IMlevel. As was noted above, the IM binding response level is preferablydetermined at multiple different time occasions, starting e.g. frombefore the cancer treatment and continuing during and after thetreatment.

The normal or healthy subject IM binding response level (concentrationor amount) to be compared with the measured IM level can be determinedby measuring the IM binding response level in a series of samples,preferably body fluid samples, e.g. blood serum samples, from healthysubjects. These healthy subjects preferably do not have or have had anydemonstrable tumors, i.e. exhibit no clinical sign of tumor or cancerdisease. The IM binding response level of these healthy control subjectsmay be determined using the same methods as for the determination of thebinding response level in the subject under investigation, as isdiscussed in more detail below. In an embodiment, the normal IM bindingresponse level is the average of the IM binding response levels measuredfrom the series of healthy subject samples.

The IM concentration level in healthy human subjects is about 1 pM orless, whereas for human tumor patients the level increases to severalhundred pM. A determined IM concentration level of 2 or 3 pM or abovecould be determined as an abnormal level, and thus may correspond toestimating a likelihood of tumor disease progression. However, theconcentration level that corresponds to a likelihood of cancer diseaserelapse may be different for different types of tumors and differentkinds of subjects.

Prior art prediction methods, as exemplified by the U.S. Pat. No.5,698,409, are, as was briefly mentioned in the background section,based on measurement of the enzyme activity of TK (and TK1) in serumsamples. In the above-identified patent, O'Neil uses monoclonalantibodies that binds to the enzymatically active tetrameric 100 kDaform of TK1, probably to the active site of the tetrameric 100 kDa formof TK1, and inhibits the TK1 enzyme activity. In order to predictrecurrence of a tumor based on the TK enzyme activity O'Neil presumesthat there is a direct correlation between the (serum) TK enzymeactivity and the level (concentration) of TK1 comprising immunoreactivematerial. As is shown by the present invention, such a correlationexists for healthy human individuals, for patients with certainnon-cancer diseases, e.g. kidney malfunction, hemorrhage and lunginfection. A correlation between TK serum activity and concentration canalso be found for a few cancer types before the cancer treatment.However, no such a correlation has been found during or, in particular,after cancer treatment. A possible explanation for this lack ofcorrelation between concentration of serum TK1 (STK1) and the enzymeactivity of serum TK (STK) in cancer patients could be that theconcentration of STK1 and the activity of STK reflect differentsubpopulations of thymidine kinase. In addition, there may be regulatingfactor(s) in serum controlling the concentration of TM/STK1 and/orenzyme activity of STK. Furthermore, the TK enzyme activity is alsoknown to be very sensitive to changes in pH and temperature.

Thus, those prior art methods that determines likelihood of tumorrecurrence based on measurement of STK enzyme activity (i.e. based on ameasure in an enzyme activity domain) after cancer treatment, e.g. byusing the monoclonal antibodies disclosed in the U.S. Pat. No.5,698,409, and the presumption of a correlation between binding response(concentration) level of STK1 and STK enzyme activity give incorrectpredictions, particular in cases of solid tumors.

However, by determining the binding response level or concentration ofIM (STK1) using the antibodies discussed in this invention andestimating progress of the cancer disease based directly on the measuredbinding response level or concentration (i.e. a measure in the bindingresponse or concentration domain), a much more accurate tumor diseaseprediction is obtained.

The prediction of cancer progression according to the present inventionis based on the binding response (concentration or amount) level of IM,such as concentration of the TK1 protein, in a biological sample,preferably a body fluid sample, e.g. blood serum sample, from a subject.The binding response level of IM in the (serum) sample can be determinedin several different ways known to the person skilled in the art usingdifferent ligands. In a presently preferred embodiment of the invention,the IM binding response level is determined by contacting the samplewith special TK antibodies and then measuring the antibody binding. Thisantibody binding can be measured by several methods known in the art.The IM binding response level is then preferably related to a chemicallydefined standard. In a presently preferred embodiment the standard ishuman TK1, e.g. recombinant, isolated and substantially purified humanTK1 (rhTK1), which may prepared as described by Wang et al. [Wang,Biochemistry, 38:16993, 1999]. Thus, the same anti-TK antibody (ligand)that is used in the sample from a subject to be treated for or alreadytreated for cancer is used in test samples comprising different knownconcentrations of rhTK1. By determining the antibody binding inrespective test sample a standard curve of the correlation betweenantibody binding and concentration level is obtained. The IMconcentration can then be determined from the measured antibody bindingand the standard curve. An alternative relation between theconcentration level and binding response level could be a mappingfunction that uses a determined IM binding response measure as input andoutputs a corresponding concentration value. Such a function can bedetermined using the standard curve and/or the rhTK1-including testsamples.

The assay method used for antibody binding has to be sensitive enough todetect the low amount of IM in subjects, especially in healthy subjects.The assay method is preferably sensitive enough to detect IMconcentration levels of at least 1 pM, preferably at least 0.7 pM, morepreferably at least 0.5 pM, such as at least 0.3 pM. An example of sucha sensitive assay method is enhanced chemoluminescence (ECL) dot blotassay. As is known in the art, the procedure of ECL dot blot assay issubstantially the same as for Western blotting. Basically, blood serum(or another body fluid) is applied onto a membrane e.g. a nitrocellulosemembrane. After (optional) blocking, primary (anti-TK1) antibodies areadded, followed by biotinylated secondary antibodies. The membrane isthen immersed in a buffer solution with H₂O₂ followed by incubation witha buffer solution with avidin-HRP-streptavidin (avidin-Horse RadishPeroxidase-streptavidin) and/or streptavidin-HRP. The light is thendetected by e.g. a digital CCD (Charged Coupled Device) camera orexposed to X-ray film. The ECL dot blot assay is able to detectconcentration levels of less than 0.3 pM [He, Internal. J. Biol. Marker,15:139, 2000].

Another possible assay method is RIA (Radio Immune Assay), where(anti-TK1) antibodies are labeled with an isotope, e.g. ¹²⁵I. Althoughthis method is as sensitive as ECL dot blot, the used ¹²⁵I is anunstable isotope with a half life of a few weeks. In addition expensivedetection equipment (γ-scintillation counter) is usually required fordetermination of antibody binding.

The antibodies used in the above-discussed IM assay method could bepolyclonal and/or monoclonal antibodies that bind specifically to anepitope of the TK1 protein, preferably TK1 protein from human subjectsand more preferably both enzymatically active and inactive forms of thehuman TK1 protein. The epitope is preferably (physically) separated fromthe active site of TK1, i.e. the antibodies should not substantiallyaffect or inhibit the enzyme activity of TK1 by direct binding to theactive site. According to the invention, the antibodies preferably bindspecifically to the C-terminal portion of TK1 protein and morepreferably to the sequence of TK1 protein from amino acid 179 to aminoacid 234, or a portion thereof, e.g. a sequence from amino acid 195 toamino acid 225, such as a sequence from amino acid 210 to amino acid224, or a polypeptide sequence comprising the amino acid sequence of TK1protein from amino acid 179 to amino acid 234. Examples of suchantibodies are described in the U.S. Pat. No. 6,083,707, the teaching ofwhich is hereby incorporated by reference in the present invention. Inthis US Patent, polyclonal antibodies against a 15 amino acids(KPGEAVAARKLFAPQ, SEQ ID NO: 1 or Acetyl-KPGEAVAARKLFAPQ, SEQ ID NO: 2)long sequence of the C-terminal end of TK1 are produced. The antibodiesof the invention are, thus, generated to surface exposed parts(epitopes) of TK1 protein. By using an antibody against one of thesequence discussed above, the binding response level and concentrationlevel of TK1 protein and possibly TK1 complexed with other moleculescould be determined. This means that the antibodies of the inventionpreferably determines a total amount of the TK1 protein, includingenzymatically active and inactive forms of the TK1 protein and the TK1protein complexed to other molecules, which include the epitopes thatthe antibodies have affinity for.

Blocking agents in certain sera might prevent binding of the antibodiesto TK1 protein by, for example, blocking the epitope to which theantibody binds and/or changing, upon binding TK1 protein so that theantibody binding epitode is moved from an accessible (surface) locationto a location where it is inaccessible for the antibody.

In a particular embodiment, the prediction method of the invention is adecision support method, i.e. a non-diagnostic method. This means thatthe decision support method will result in decision support informatione.g. as exemplified by a binding response, concentration or likelihoodvalue, which is merely interim results. Additional data and thecompetence of a physician are typically required for providing a finaldiagnosis. Furthermore, other parameters than the IM binding responselevel (IM concentration level) may influence the progression of a tumordisease, such as the case history, age and sex of the subject, the typeof tumor, genetic factors etc. Thus, the invention gives a decisionsupport upon which a physician can base his decision about whichmeasures that should be taken.

The present invention is applicable on several different types ofcancers, including, but not limited to, human sarcomas and carcinomas,e.g. fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, enotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyo sarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung cacinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, hemangioblastoma, oligodendroglioma,melanoma, neuroblastoma and retinomblastoma, leukemias, e.g. acutelymphocytic leukemia (ALL), and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erytholeukemia), chronicleukemias (chronic myelocytic leukemia, chronic granulocytic leukemiaand chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenström's macroglobulinemia and heavy chain disease. The presentinvention is in particular applicable in prediction progress of cancerswith solid tumor, e.g. breast and gastric cancer.

The tumor monitoring and prediction method of the invention issummarized in FIG. 7. Starting with step S1, a binding response level ofan immunoreactive material (IM) comprising (enzymatically active andinactive) TK1 protein is determined in a sample, preferably body fluidsample, e.g. blood serum sample, from a subject, preferably mammaliansubject and more preferably human subject, treated for cancer. Note thatthe cancer treatment does not have to be completed when the taking ofsample from the subject is performed. The IM binding response level isdetermined using an ligand, preferably an antibody that bindsspecifically to the TK1 protein, more preferably an antibody that bindsspecifically to the C-terminal portion of TK1 protein. In step S2, theestimation of the likelihood of progress or relapse of the cancerdisease, e.g. as manifest by local and/or metastatic tumor recurrence orappearance, is performed based directly on the determined IM bindingresponse level. This likelihood may be estimated based on a comparisonof the detemlined IM binding response level with an IM binding responselevel as determined earlier for the subject or from healthy subjects.

According to another aspect of the invention there is provided a systemfor predicting progression of a cancer disease in a subject treated forcancer. An IM binding response level, or concentration level of IM, e.g.concentration of serum TK1 protein, is determined in the subject, e.g.by one of the assay methods discussed in the foregoing. The determinedbinding response level or concentration is then the input data in thesystem, preferably together with a IM binding response (concentration)level of healthy individuals and/or an earlier determined bindingresponse level for the subject, in order to obtain an estimation of thelikelihood of cancer relapse in the subject.

The system could be a computer system with dedicated software adaptedfor tumor progression prediction or a neural network. Severalmeasurements of IM level from subjects treated for cancer and obtainingsubsequent tumor progression and relapse and not obtaining tumorrelapse, respectively, and (normal) IM binding response level fromhealthy subjects may be used for designing such a software. Baseddirectly on these measurements, the software could be able to provide anestimation of tumor progression and relapse from IM binding responselevels.

The system could also require additional input parameters for a moreaccurate estimation, such as additional case history parameters, age andsex of the subject, cancer type, genetic markers, other biochemicalparameters, etc. The system or dedicated software of the system could beconfigured for using an equation of formula with IM binding responselevel or IM concentration (and possible other parameters) as input inthe equation. The software/system then calculates the likelihoodestimation. Alternatively, one or several cutoff limits could beemployed for determination of the estimation. Thus, estimating thelikelihood of tumor recurrence based directly on a determined IM bindingresponse or concentration value from a subject also anticipates that thedetermined IM binding response values (concentrations) are input in some(basic) function, possible with other input parameters. The basic ideais that the estimation is based directly on measures of a bindingresponse (concentration) domain and does not require a mapping from thisbinding response domain to an enzyme activity domain for performing theestimation.

The system could also incorporate equipment for determination of the IMbinding response level in a subject's sample. In such a case, a bodyfluid sample is input in the system, which determines the IM level andcalculates the tumor progression and relapse likelihood estimationdirectly based on the IM level. Such a binding response determiningequipment could be based on one of the assay methods, i.e. ECL dot blotor RIA with anti-TK1 antibodies, discussed in the foregoing.

EXAMPLES ECL Dot Blot Assay

The procedure of the ECL dot blot assay is the same as for Westernblotting, with slight modification [He, Internal. J. Biol. Marker,15:139, 2000]. Three μl of serum of venous blood from non-heparinisedblood tubes was directly applied onto a nitrocellulose membrane(HybandTM-C, Amersham). The blood samples were taken in the morningbetween 7-9 AM of persons who did not take any breakfast. The drawnvenous blood in the non-heparinised tubes can be stored for at least 24hours at +4° C., or centrifuged at 1,500 g for 5 min and then stored at−20° C. for 5 years and at −80° C. for more than 10 years. Recombinanthuman TK1 (rhTK1) was used as a standard, as initially described by He,et al. [He, Europ. J. Cell Bio., 70:117, 1996] and modified as describedby Wang, et al. [Wang, Biochemistry, 38:16993, 1999]. The employedmembrane was blocked in TBS (Tris-Buffered Saline) buffer with 10%non-fat milk for 4 hours and then primary (anti-TK1) antibody was addedand incubated at +4° C. over night or at room temperature for two hours.After incubation with a, biotinylated secondary antibody for one hour atroom temperature, the membrane was immersed in TBS buffer with 3% H₂O₂for 5 min. The membrane was then incubated in TBS buffer withavidin-HRP-streptavidin and exposed to X-ray film. The intensity of asingle spot on the film was determined by a Laser Densitometer. From theintensity of the rhTK1 of known concentrations, the intensity of the IMwas recalculated and expressed as pM.

Characterization of Anti-TK1 Antibodies

Anti-TK1 antibodies were characterized by Western blot, isoelectricfocusing, immunoprecipitation and immunohistochemistry. The results aresummarized in Table 1 below. According to the Western blots, themonoclonal antibodies against the 15 amino acids long peptide sequence(SEQ ID NO: 2) (mAb1D11 and mAb1E3) or rhTK1 (mAb26-3 and mAb26-5)recognize the native form of the TK1 protein in human tumor cells, aswell as the TK1 protein in serum of patients with malignancies, but notthe denatured 25 subunit of TK1 protein. No band in the electrophoresegel was found in the TK1 negative cells, in serum of healthy persons orin the presence of competing antigen. Immunoprecipitation testsindicated that mAb1E3, although not directed to the active site of theTK1 protein, affects the enzyme activity of TK1. mAb26-3+5 and mAb1D11inhibit TK1 activity in the supernatant by making an immunocomplex withthe TK1 protein, which is collected in the pellet. Isoelectric focusingshowed that the TK1 protein has a pI value of ≈7.0, when blotted withmAb26-3+5. However, no band was detected when blotted with mAb1D11 andmAb1E3. The polyclonal rabbit anti-TK1 antibody (pAb) recognizes thesubunit 25 kDa in SDS gelelectrophorese and gives a pI value of TK1 of≈8.3. The chicken IgY anti-TK1 antibody (SSTK Biotech Inc., China),directed against a 31 amino acids sequence or amino acids 194 to 225 ofthe TK1 protein, recognize both the native form of TK1 and the subunit25 kDa and give a pI value of ≈8.3. Finally, monoclonal antibodies (mAbUSA) against the whole TK1 protein (QED Bioscience Inc, San Diego, USA)recognize the 25 kDa subunit of TK1.

TABLE 1 Western blot of TK1 in CEM cell lysates TK1 in Native IEFImmuno. serum SDS gel gel gel TK1 activity staining Native 25 kDa Rm0.16 pI In pellet In sup. Cytoplas. gel, Ab Antigen TK1 TK1 TK1 TK1 TK1TK1 cancer CEM cells + − + − + − + − + − + − patient Comments mAb26-3rhTK1 − − + − 7.0 − + − − − + − 7.0 Some cross-section in mAb26-5 rhTK1− − + − 7.0 − + − − − + − 7.0 malignant and normal tissue mAb26-3 + 5rhTK1 − − ++ − 7.0 − ++ − − − + − Rm 0.31 Can be used for STK1 mAb USATK1 + − + − Can be used for STK1, some cross-reactions in healthy serummAb1D11 15 aa − − ++ − − − ++ − − − + − Rm 0.31 Some cross-reactions,can be mAB1E3 15 aa − − + − − − − − − − + − Rm 0.31 used for STK1 afterpurification pAb 15 aa + − + − 8.3 − ++ − − − ++ − 7.0 Weakcross-reactions Chicken IgY 31 aa + − + − 8.3 − + − Good for STK1

All the antibodies discussed above and presented in Table 1 can be usedfor immunocytochemical staining, both in cell lines and in paraffinembedded material. The anti-TK1 antibody reaction was localized to thecytoplasm of tumor cells. No or very little staining in mutant cellslacking TK1 enzyme activity was found, as well as in restinglymphocytes. Thus, the anti-TK1 antibodies tested can be used todetermine the IM binding response level and concentration. Theseantibodies also recognize different epitopes on the TK1 protein,probably identifying various subpopulations of TK1.

IM Concentration and STK Enzyme Activity in Healthy Subjects

IM (STK1) concentration as measured by the above-discussed dot blot ECLimmunoassay was compared to STK enzyme activity as determined by the RIAassay in healthy persons and the results are presented in FIG. 1 andTable 2. Both the IM concentration and the STK enzyme activity were low(less then 2 pM and 2 U/L, respectively). There was a significantcorrelation between IM concentration and STK enzyme activity (r=0.67,p<0.01) for these healthy persons.

The IM concentration and STK activity were also determined in patientswith non-cancer diseases. In six of 19 such patients with kidneymalfunction, hemorhage, cirrhosis of liver and lung infection andnon-infection diseases, IM concentration and STK activity were elevatedby 1.6 and 1.8 times, respectively, as compared to the healthy controls.In one case of hemorhage and one case of liver cirrhosis, the IMconcentration was 5 times higher and the STK activity 2-4 times abovethe cut-off values of 2 pM and 2.4 U/L, respectively. There was asignificant correlation between the IM (STK1) concentration and the STKactivity in patients with these types of non-cancer diseases (Table 2).

In Table 2, correlation and its significance values are based onPearson's correlation coefficients.

TABLE 2 STK1 concentration versus STK activity Tumor type n r_(p) pHealthy 43 0.67 <0.01 Non-cancer diseases 19 0.72 <0.01 Benign tumor 340.63 0.01 < p < 0.05^(a) Leukemia 25 0.44 0.01 < p < 0.05^(a) Gastric109 0.36 <0.01 Colon 17 −0.21 no Rectum 28 0.30 no Breast 22 0.58 <0.01Lung 39 −0.15 no Lymphoma 6 0.00 no Hepatoma 12 0.26 no Brain 8 −0.26 no^(a)Significance values are less than 0.05 but larger than 0.01.

IM Concentration and STK Activity in Tumor Patients Before Treatment

IM (STK1) concentrations from 752 tumor patients with 9 different typesof malignant tumors were determined by the ECL dot blot immunoassay andwas found to be increased up to 200 times, as compared to the healthypersons. IM concentrations were also measured in serum of patients withmorphological benign lesions. The results of the measured concentrationsare presented in FIG. 2, where A represents leukemias, B representsgastric tumors, C represents colon tumors, D represents rectum tumors, Erepresents breast tumors, F represents lung tumors, G representslymphomas, H represents hepatomas, I represents brain tumors, Jrepresents other types of malignant tumors, K represents morphologicalbenign lesions and L represents healthy persons. In the patients withmalignant tumors the concentration of IM was found from almost normalvalues (cut-off 2 pM) (29/752) to 100 pM (711/752), and in some cases upto 200 pM (13/752). Thus, about 95 percent of the patients withmalignancy show IM concentrations above a cutoff value of IM of 2 pM.

STK enzyme activity was also determined in parallel in 264 patients withtumor, benign lesions, non-cancer diseases and healthy persons. Whilethe concentration of IM varied extensively between the various patients,STK activity showed only a limited variation. In benign lesions,leukemia, breast cancer and gastric cancer, significant correlationbetween IM concentration and STK activity were found for the patientsbefore any cancer treatment (see Table 2). However, no correlation wasfound for the majority of the investigated tumor types in the untreatedindividuals, see FIG. 4.

IM (STK1) concentration was also investigated in 68 preoperativepatients with cancer of the gastro-intestinal track with regard to thepresence or absence of metastasis (no metastasis (M0) n=46, andmetastasis (M1) n=22) and the degree of differentiation (high ormoderate high differentiation n=56, and low differentiation n=12)according to guidelines set out in the clinical TNM (UICC, Fifthedition, 1997). The results are summarized in Table 3. The IMconcentrations were 4.5 times higher in the patients with metastasis(M1), as compared to patients without metastasis (M0) (p<0.01). Theconcentration in the patients with low differentiated tumors, was alsohigher, as compared to patients with high/moderate high differentiatedtumors, although not significantly different (p>0.05).

TABLE 3 No. IM conc. (pM) t value p value Differentiation 68 High ormiddle 56 38 + 70 0.88 >0.05 Low 12 57 + 67 Metastasis 68 Yes (M1) 22 87 + 106 4.20 <0.001 No (MO) 46 19 + 20

Gastric Cancer

IM (STK1) concentrations and STK enzyme activities were determined in 43patients with gastric cancer (GC) by means of the dot blot ECLimmunoassay and by the RIA-activity assay, respectively. FIG. 3illustrates a Western blot and dot blot from one example of a tumorpatient (P3), a dot blot of a healthy individual (healthy) and ofdifferent concentrations of human recombinant TK1 (rhTK1). Thedetermined STK1 concentration and STK activity values of preoperativepatients are presented in Table 4. The IM concentration and the STKactivity were significant elevated in the tumor patients beforeoperation.

TABLE 4 2-tailed p between GC Linear correlation IM conc. STK activityand healthy sera coefficients between IM (PM) (U/I) IM STK activityconc. and STK activity Preoperative 27.7 +26.7 5.9 + 4.6 0.012 0.0003 r= 0.36 GC (n = 43) Healthy (n = 43) 0.98 + 0.4  1.1 + 0.4 r = 0.33

Thirty-five days after operation the IM concentrations decreasedsignificantly by about half in tumor free cases, (p=0.0106), while theSTK enzyme activities did not. In the patients with metastasis (M1), IMconcentration increased further to 173% 35 days after operation. No suchincrease was seen in STK enzyme activity, see Table 5. The unchanged IMconcentration in M0 patients at day 7 after operation is partly due toan increase in the IM concentration in connection with operation trauma(data not shown).

TABLE 5 2-tailed p between 0-day Spearmen's and 7-day or 35-daycorrelation coeff. IM conc. STK activity postoperative sera between IMconc. (pM) (U/l) IM STK activity and STK activity  0 day M0 (n = 8)18.21 ± 12.7  11.1 ± 3.8 R = 0.833 p = 0.01 M1 (n = 6) 31.6 ± 29.9  5.0± 2.1 R = 0.371 p = 0.468  7 days M0 (n = 8) 19.9 ± 10.3 12.3 ± 3.10.0751 0.0741 R = 0.81 p = 0.015 M1 (n = 6) 30.7 ± 30.5  4.7 ± 1.90.0672 0.107 R = 0.37 p = 0.968 35 days M0 (n = 8) 9.60 ± 9.0  10.2 ±5.6 0.0106 0.797 R = 0.61 p = 0.072 M1 (n = 6) 54.9 ± 17.1  4.6 ± 2.20.0605 0.329 R = 0.812 p = 0.042

As is evident from Tables 4 and 5, the IM concentrations can be used tomonitor the results of the treatment in gastric cancer, in contrast toSTK enzyme activity. Furthermore, the IM concentration level, but notthe STK activity, is able to detect relapse of the cancer disease.

Leukemias

IM (STK1) concentration as measured by the dot blot ECL immunoassay andSTK enzyme activities measured by RIA assay were determined in 24patients with leukemia. There was a significant correlation betweenthese two parameters in the patients before start of the treatment(r=0.44), see diagram A in FIG. 4.

Of these 24 patients 6 were followed during and after treatment. Therewas a reduction in IM concentration in those patients receivingchemotherapy. When the chemotherapy was ended, the IM concentrationincreased. The STK enzyme activity was still high after the start oftherapy, which indicates that the concentration of IM is a more reliableassay to predict the outcome of the treatment as compared to the STKactivity.

Breast Cancer

The IM (STK1) concentrations in serum in a cohort of 120 breast cancerpatients, included in a controlled clinical trial of adjuvant endocrinetherapy, were determined in 67 of these patients three month afteroperation, using the anti-TK1 chicken IgY antibody (see Table 1). The IMconcentration at three months was related to that obtained at 21 daysafter operation. Twelve patients developed distant metastatic cancerdisease and 7 loco-regional tumors as the first event during a medianfollow-up time of 11.6 years.

The 67 patients were subdivided into three equally sized groups ofpatients with a relative IM concentrations of <0.78, 0.78-1.08 or >1.08,as determined by dividing the IM concentration at three months aftersurgery with the corresponding IM concentration 21 days after operation.A multivariate analysis of patients with any tumor recurrences (distant-or loco-regional), considering nodal- and ER-status, tumor size,endocrine treatment and age, showed that the patients with a relative IMconcentration >1.08 were statistical significant different from thosepatient who have a relative IM concentration of <0.78 (p=0.004). Thehazard rate ratio for developing any recurrences in the patients with arelative IM concentration >1.08 was about 6-7 times higher than patientswith the relative concentrations <0.78.

TABLE 6 Unadjusted effect Adjusted effects^(a) Type of 1^(st) eventHazard Hazard Events/ rate ratio rate ratio IM ratio ^(b) Patients (95 %CI^(d)) P-value^(c) (95 % CI^(d)) P-value^(e) Distant recurrence <0.782/22 1 1 0.78-1.08 3/22 1.5 (0.3-9.0)  1.2 (0.2-7.3)  >1.08 7/23 3.8(0.8-18.1) 0.056 2.5 (0.5-12.1) 0.19 Any recurrence^(e) <0.78 2/22 1 10.78-1.08 5/22 2.6 (0.5-13.6) 2.3 (0.4-12.3) >1.08 12/23  7.7 (1.7-34.5)<0.001 6.1 (1.3-28.5) 0.004 ^(a)Adjusted for age (<45 years, ≥45 years),nodal status (pNO, pN+), tumor size (<20 mm, ≥20 mm), ER status (ER−,ER+) and endocrine treatment (yes, no). Seven patients are excluded fromthis analysis due to missing information on ER status. ^(b)Ratio betweenIM concentration measuredat 3 months and 3 weeks after operation. Cutoff points were obtained by dividing the IM distribution into 3 equallysized groups. ^(c)Test for trend. ^(d)Confidence interval.^(e)Loco-regional or distant reccurence.

The estimated cumulative incidence rates of any recurrence at 5 yearswere 0.09 (95% CI: 0.02-0.34), 0.23 (95% CI: 0.11-0.49) and 0.48 (95%CI: 0.31-0.73) for the relative STK1 concentrations <0.78, 0.78-1.08and >1.08, respectively, see FIG. 5. Thus, a high IM concentration 3months after the cancer treatment, as represented by the group >1.08, isa clear indication of a high likelihood of (loco-regional or distant)tumor recurrence.

IM (STK1) concentration and STK enzyme activity were determined in 37 ofthe patients. These results were also compared with CA 15-3determinations, which is regarded as “the golden marker” within thetumor marker field. FIG. 6 illustrates the determined IM concentration,STK activity and CA 15-3 concentration at 3 months after operationexpressed as percentage of corresponding values 21 days after operation.In 15 patients (A) there was a relapse of tumor within 1 to 5 yearsfollowing the operation, whereas there was no tumor relapse for 22patients (B). As is shown in the figure, already at 3 months afteroperation the IM concentration was markedly higher in 50-60% of thosepatients (A) that 1 to 5 years later again developed tumors. However,there was no difference in STK activities or CA 15-3 values in thepatients with (A) or without (B) any recurrent tumors. The IMconcentration also predicted 25-30% of those patients (B) who did notsubsequently get recurrent tumor disease.

Thus, there is concluded that there is a significant trend toward higherrelative IM concentrations in patients developing distant and/orloco-regional recurrent cancer disease, during the follow up time of11.6 years. Such information can be used to avoid, prolong or changeadjuvant therapy due to an improved risk assessment.

It will be understood by a person skilled in the art that variousmodifications and changes may be made to the present invention withoutdeparture from the scope thereof, which is defined by the appendedclaims.

What is claimed is:
 1. A method of selecting treatment for a subject diagnosed with a cancer disease, the method comprising the steps of: contacting an antibody that binds specifically to thymidine kinase 1 (TK1) protein with a first body fluid sample taken from the subject before completion of a cancer treatment selected from the group consisting of surgery, radio therapy, chemotherapy and a combination thereof; determining a first amount of antibody binding to the TK1 protein in the first body fluid sample; correlating the first amount of antibody binding to a first concentration of TK1 protein in the first body fluid sample using a standard curve defining a correlation between an amount of antibody binding to recombinant human TK1 (rhTK1) and a concentration of rhTK1; contacting the antibody with a second body fluid sample taken from the subject within one to six months after completion of the cancer treatment; determining a second amount of antibody binding to the TK1 protein in the second body fluid sample; correlating the second amount of antibody binding to a second concentration of TK1 protein in the second body fluid sample using the standard curve; identifying the patient as having a high risk of future cancer relapse if the second concentration is equal to or higher than the first concentration and otherwise identifying the patient as having a low risk of future cancer relapse; and selecting an adjuvant treatment for the subject based on whether the patient is identified as having high risk or low risk of future cancer relapse.
 2. The method according to claim 1, wherein the antibody binds specifically to both enzymatically active and inactive forms of the TK1 protein.
 3. The method according to claim 1, wherein the antibody that binds specifically to a C-terminal portion of the TK1 protein.
 4. The method according to claim 1, wherein the antibody binds specifically to a peptide selected from SEQ ID NO: 1 and SEQ ID NO:
 2. 5. The method according to claim 1, wherein the first body fluid sample is a first blood serum sample; the second body fluid sample is a second blood serum sample; and the antibody binds specifically to both a serum form of TK1 (STK1) protein.
 6. The method according to claim 1, wherein the cancer disease is a solid tumor cancer disease.
 7. The method according to claim 1, wherein the patient is identified as having high risk or low risk of future cancer relapse without determination of an enzyme activity level of TK1 in the first body fluid sample or the second body fluid sample. 